Peptides As Therapeutics with Enhanced Bioactivity

Send Orders of Reprints at [email protected] Current Medicinal Chemistry, 2012, 19, 4451-4461 4451 Peptides As Therapeutics with Enhanced Bioactivity D. Goodwin1, P. Simerska1 and I. Toth*,1,2, 1The University of Queensland, School of Chemistry and Molecular Biosciences; 2School of Pharmacy, St. Lucia 4072, Queensland, Australia Abstract: The development of techniques for efficient peptide production renewed interest in peptides as therapeutics. Numerous modi- fications for improving stability, transport and affinity profiles now exist. Several new adjuvant and carrier systems have also been developed, enhancing the immunogenicity of peptides thus allowing their development as vaccines. This review describes the established and experimental approaches for manufacturing peptide drugs and highlights the techniques currently used for improving their drug like properties. Keywords: Peptide, drug delivery, vaccine, manufacture, bioavailability, peptide therapeutic, immunogenicity, peptide drug, peptide synthesis, clinical trials. INTRODUCTION side-chain reactivity, degree of modification, incorporation of un- natural components, in addition to the required purity, solubility, Natural and synthetic peptides have shown promise as pharma- stability and scale. There are two strategies for peptide production - ceutics with the potential to treat a wide variety of diseases. This chemical synthesis and biological manufacturing. potential is often overshadowed by the inability of the peptides to reach their targets in an active form in vivo. The delivery of active Chemical Peptide Synthesis peptides is challenging due to inadequate absorption through the Chemical synthesis has been used for the production of peptides mucosa and rapid breakdown by proteolytic enzymes. Peptides are in both research and industry and led to the development of the usually selective and efficacious, therefore need only be present in majority of peptide drugs [2]. These chemical-based strategies were low concentrations to act on their targets. The metabolism of pep- usually favored due to their unique ability to incorporate any num- tides is superior to other small molecules due to the limited possi- ber of non-natural components. Two chemical methodologies- bility for accumulation and result in relatively non-toxic amino solution-phase (SPS), and solid-phase peptide synthesis (SPPS) [4] acid, peptide metabolites. These properties contribute towards the - are the predominant methods of chemical synthesis, but many overall low toxicity of peptides, with a limited risk of adverse inter- other approaches, including hybrid synthesis and chemoselective actions. ligation, have also been studied. The most important classes of peptides that have been investi- Solution-Phase Peptide Synthesis gated include insulin, gonadotropin-releasing hormone, calcitonin, the enkephalins, the glucagons, other hormone analogs, enzyme First described by du Vigneud in 1953, the SPS method was inhibitors and vaccines. The majority of peptides were targeted used for the production of most commercial peptide therapeutics towards treatment of cancers and metabolic disorders, with a large [5]. The method was based on the coupling of peptide fragments or number targeting G-protein coupled receptors. In 2010, there were a single amino acid in solution Fig. (1). The main advantage of SPS over 50 peptide drugs approved for marketing, with annual global was economic because of the use of relatively inexpensive building sales nearing/surpassing US$ 1 billion for the following peptide blocks and reagents. Each intermediate could be analyzed and puri- drugs: ciclosporin (e.g. Neoral®, Novartis), goserelin acetate (Zo- fied, leading to products of higher purity and easier final purifica- ladex®, AstraZeneca), glatiramer acetate (Copaxone®, Teva Phar- tions. The main limitation of SPS was the need to modify each maceuticals), leuprolide acetate (e.g. Lupron®, Abbott Laborato- intermediate reaction step to synthesize a required peptide thus, the ries), and octreotide acetate (Sandostatin®, Novartis). Additionally, development of each synthetic scheme was long, laborious, and in 2010, more than 100 peptide drug candidates were reported to be relatively expensive. Peptide pharmaceuticals from as few as 3 in clinical development [1]. amino acids (e.g. thyrotropin-releasing hormone) to peptides of 32 residues (e.g. calcitonin) have been synthesized for clinical use This review describes novel approaches in peptide synthesis using SPS [2]. and the use of peptides as therapeutics. Solid-Phase Peptide Synthesis PEPTIDE THERAPEUTICS Ten years after the development of SPS, Merrifield introduced SPPS [6]. This method was based on the coupling of protected Initially, inefficient and expensive manufacturing processes amino acids to an insoluble support Fig. (1). The simplicity of this hampered the development of peptide-based therapeutics. Neverthe- process was compatible with automated synthesis for the large scale less, the production of the peptide drug enfuvirtide (Fuzeon®, production of peptides. SPPS proved to be faster and less laborious Roche, a human immunodeficiency virus (HIV) membrane fusion than SPS. Two forms of SPPS methods were developed based on inhibitor), demonstrated that preparing a 36-amino acid peptide on the protection of the -amino group of an amino acid, the tert- a ton-scale annually, could be cost effective [2]. butyloxycarbonyl (Boc) group was deprotected by acid (e.g. The establishment of industrial-scale peptide manufacturers trifluoroacetic acid) [7], while 9-fluorenylmethyloxycarbonyl meant that the chemical industry had to supply increased demands, (Fmoc) group was removed by base (e.g. piperidine) [8]. which caused a decrease in prices, increased availability of amino The most important limitation to the use of SPPS on an indus- acids and consequently, market growth [3]. trial scale was the requirement for excess starting material [2]. The methods used for the preparation of peptide therapeutics However, several peptide therapeutics such as ziconotide (Prialt®, have depended on a number of important criteria: sequence length, Elan), exanatide (Byetta®, Amylin/Eli Lilly Co.), pramlintide (Symlin®, Amylin) and degarelix (Firmagon®, Ferring) were syn- *Address correspondence to this author at the School of Chemistry and Molecular thesized by SPPS and approved by the U.S. Food and Drug Biosciences, The University of Queensland, St Lucia, Queensland, Australia; 4072, Administration (FDA) [9, 10]. Since 1992, microwave irradiation Tel: +61 7 33469892; Fax: +61 7 33654273; E-mail: [email protected] has been used extensively to accelerate SPPS by providing a con- 1875-533X/12 $58.00+.00 © 2012 Bentham Science Publishers 4452 Current Medicinal Chemistry, 2012 Vol. 19, No. 26 Goodwin et al. venient rapid heating method [11]. SPPS was used to make Grami- Pseudo-proline (Pro) modified amino acid residues were cidin A (peptide antibiotic) and CSF114(Glc) (a glycopeptide im- designed to minimize aggregation and racemization when synthe- munological probe) with a good yield and high purity [12]. sizing difficult peptide sequences [18]. Pseudo-prolines (Pro) are Hybrid Synthesis serine- or theonine-derived oxazolidines or cysteine-derived thio- zolidines with proline-like ring structures Fig. (2). A fragment con- Due to the time limitations and adverse side-reactions present in densation of the complex RNase1-39 glycopeptide made with Pro traditional SPS and SPPS protocols, synthesis of some peptide fragments was carried out successfully using SPPS without racemi- sequences has often proven difficult. Hybrid synthesis, which com- zation [19]. The use of polyethylene glycol (PEG)-based resins, bines SPS and SPPS, merged the benefits of both protocols Fig. (1) particularly ChemMatrix® (CM), showed benefits over traditional [4]. The production of enfurvirtide, established using SPPS, was polystyrene-based resins [20, 21]. CM was very stabile with a high changed to a hybrid strategy for manufacture in the scale of metric loading capacity and has been used in both aqueous solutions and tons annually from three or more fragments [2]. during the synthesis of difficult hydrophobic peptides [21]. The use Chemoselective Ligation of Pro in combination with CM was successful in the synthesis of a 68-amino acid chemokine, which was unsuccessful using a poly- Another option for the elongation of peptide chains is through styrene resin due to aggregation [22]. Kessam et al. suggested that a chemoselective ligation techniques, which offer the ability to cou- newly developed resin incorporating PEG polymerized onto a ple unprotected peptide fragments Fig. (1). Native chemical ligation poly(vinyl alcohol) core may be even more valuable, economically was described as one example of chemoselective ligation, which, and environmentally, after demonstrating its promise against other through a thiol exchange and spontaneous rearrangement, ulti- resins in synthesis of a section of acyl carrier protein [23]. mately formed natural peptide bonds between peptide fragments [13]. The limitation of this strategy was the requirement of an N- terminal cysteine and C-terminal thioester. A modified method used NH O auxillary thiols in place of the cysteine residues, which were HN removed at the site of ligation and allowed the formation of a non- R4 N cysteine linkage [14, 15]. R3 O Improving Chemical Synthesis R 2 X H Due to ecological concerns about the impact of chemical syn- R1 thesis, there has been pressure

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